One mechanism for purifying, separating, or concentrating molecules of interest is called Synchronous Coefficient Of Drag Alteration (or “SCODA”) based purification. SCODA, known in some embodiments as scodaphoresis, is an approach that may be applied for purifying, separating, or concentrating particles. SCODA may be applied, for example, to DNA, RNA and other molecules including proteins and polypeptides.
SCODA based transport is used to produce net motion of a molecule of interest by synchronizing a time-varying driving force, which would otherwise impart zero net motion, with a time-varying drag (or mobility) alteration. If application of the driving force and periodic mobility alteration are appropriately coordinated, the result is net motion despite zero time-averaged forcing. With careful choice of both the temporal and spatial configuration of the driving and mobility altering fields, unique velocity fields can be generated, in particular a velocity field that has a non-zero divergence, such that this method of transport can be used for separation, purification and/or concentration of particles.
SCODA is described in the following publications:                U.S. Patent Publication No. 2009/0139867 entitled “Scodaphoresis and methods and apparatus for moving and concentrating particles”;        PCT Publication No. WO 2006/081691 entitled “Apparatus and methods for concentrating and separating particles such as molecules”;        PCT Publication No. WO 2009/094772 entitled “Methods and apparatus for particle introduction and recovery”;        PCT Publication No. WO 2009/001648 entitled “Systems and methods for enhanced SCODA”        PCT Publication No. WO 2010/051649 entitled “Systems and methods for enhanced SCODA”;        PCT Publication No. WO 2010/121381 entitled “System and methods for detection of particles”;        Marziali, A.; Pel, J.; Bizotto, D.; Whitehead, L. A., “Novel electrophoresis mechanism based on synchronous alternating drag perturbation”, Electrophoresis 2005, 26, 82-89;        Broemeling, D.; Pel, J.; Gunn, D.; Mai, L.; Thompson, J.; Poon, H.;        Marziali, A., “An Instrument for Automated Purification of Nucleic Acids from Contaminated Forensic Samples”, JALA 2008, 13, 40-48;        Pel, J.; Broemeling, D.; Mai, L.; Poon, H.; Tropini, G.; Warren, R.; Holt, R.; Marziali, A., “Nonlinear electrophoretic response yields a unique parameter for separation of biomolecules”, PNAS 2008, vol. 106, no. 35, 14796-14801; and        So, A.; Pel, J.; Rajan, S.; Marziali, A., “Efficient genomic DNA extraction from low target concentration bacterial cultures using SCODA DNA extraction technology”, Cold Spring Harb Protoc 2010, 1150-1153,each of which is incorporated herein by reference.        
SCODA can involve providing a time-varying driving field component that applies forces to particles in some medium in combination with a time-varying mobility-altering field component that affects the mobility of the particles in the medium. The mobility-altering field component is correlated with the driving field component so as to provide a time-averaged net motion of the particles. SCODA may be applied to cause selected particles to move toward a focus area.
In one embodiment of SCODA based purification, described herein as electrophoretic SCODA, time varying electric fields both provide a periodic driving force and alter the drag (or equivalently the mobility) of molecules that have a mobility in the medium that depends on electric field strength, e.g. nucleic acid molecules. For example, DNA molecules have a mobility that depends on the magnitude of an applied electric field while migrating through a sieving matrix such as agarose or polyacrylamide1. By applying an appropriate periodic electric field pattern to a separation matrix (e.g. an agarose or polyacrylamide gel) a convergent velocity field can be generated for all molecules in the gel whose mobility depends on electric field. The field dependant mobility is a result of the interaction between a reptating DNA molecule and the sieving matrix, and is a general feature of charged molecules with high conformational entropy and high charge to mass ratios moving through sieving matrices. Since nucleic acids tend to be the only molecules present in most biological samples that have both a high conformational entropy and a high charge to mass ratio, electrophoretic SCODA based purification has been shown to be highly selective for nucleic acids.
The ability to detect specific biomolecules in a sample has wide application in the field of diagnosing and treating disease. Research continues to reveal a number of biomarkers that are associated with various disorders. Exemplary biomarkers include genetic mutations, the presence or absence of a specific protein, the elevated or reduced expression of a specific protein, elevated or reduced levels of a specific RNA, the presence of modified biomolecules, and the like. Biomarkers and methods for detecting biomarkers are potentially useful in the diagnosis, prognosis, and monitoring the treatment of various disorders, including cancer, disease, infection, organ failure and the like.
The differential modification of biomolecules in vivo is an important feature of many biological processes, including development and disease progression. One example of differential modification is DNA methylation. DNA methylation involves the addition of a methyl group to a nucleic acid. For example a methyl group may be added at the 5′ position on the pyrimidine ring in cytosine2. Methylation of cytosine in CpG islands is commonly used in eukaryotes for long term regulation of gene expression2. Aberrant methylation patterns have been implicated in many human diseases including cancer. DNA can also be methylated at the 6 nitrogen of the adenine purine ring.
Chemical modification of molecules, for example by methylation, acetylation or other chemical alteration, may alter the binding affinity of a target molecule and an agent that binds the target molecule. For example, methylation of cytosine residues increases the binding energy of hybridization relative to unmethylated duplexes3-5. The effect is small. Previous studies report an increase in duplex melting temperature of around 0.7° C. per methylation site in a 16 nucleotide sequence when comparing duplexes with both strands unmethylated to duplexes with both strands methylated.
There remains a need for methods and apparatus capable of providing improved separation and purification of molecules, including identical molecules that are differentially modified.